The rapid developments in recombinant DNA techniques have resulted in the identification and isolation of many novel genes, some of known function and some of unknown function. Invariably there is a need to express the gene in a heterologous cell system in order to produce material for structure-function studies, diagnostic reagents such as monoclonal or polyclonal antibodies and material for in vivo activity testing and therapy.
Several alternative systems for the expression of foreign genes have been developed including systems based upon mammalian cells, insect cells, fungal cells, bacterial cells and transgenic animals or plants. The choice of expression system for a given gene depends upon the likely features of the encoded protein, for example any post-translational protein modifications needed for biological activity, as well as the objective of the study. Other important considerations for the investigator are the facilities available and the time and cost involved in generating the amounts of recombinant protein required.
The most widely used and convenient system for the production of foreign proteins remains that based on the prokaryote Escherichia coli. The advantages of this system comprise the ease of gene manipulation, the availability of reagents including gene expression vectors, the ease of producing quantities of protein (up to a gramme in simple shake-flask culture), speed and the high adaptability of the system to express a wide variety of proteins.
Expression of any foreign gene in E. coli begins with the insertion of a cDNA copy of the gene into an expression vector. Many forms of expression vector are available. Such vectors usually comprise a plasmid origin of DNA replication, an antibiotic selectable marker and a promoter and transcriptional terminator separated by a multi-cloning site (expression cassette) and a DNA sequence encoding a ribosome binding site. The method of transcriptional regulation varies between the various promoters now available (ptac, .lambda.pL, T7). The ptac and T7 expression based systems are controlled by the chemical inducer IPTG, whilst the .lambda., promoters are controlled by a temperature switch.
A problem encountered with E. coli based expression systems is the difficulty of producing material which is acceptable for therapeutic use. The use of complex media, antibiotic selection and potentially hazardous inducers such as IPTG may potentially render products such as recombinant antibody fragments produced by E. coli fermentation technology unacceptable to the regulatory authorities for clinical applications. Evidence demonstrating clearance of these agents from the final product must be provided in order to secure regulatory approval. Clearance of these agents, and especially demonstrating such clearance, is expensive. It is therefore desirable that an expression system should avoid the three above-mentioned problems.
A further problem is that proteins produced in bacterial cells are often precipitated as insoluble aggregates within the bacterial cell. This problem has been addressed in a number of ways in the prior art. For example, a large number of patent specifications teach solubilisation of aggregates by the use of chaotropic denaturants and subsequent renaturation. These procedures involve the use of expensive denaturing chemicals, are time-consuming and introduce chemical agents into the production process of which clearance demonstration will be required by the regulatory authorities if the product is destined for clinical use.
An alternative approach has involved attempting to secrete the heterologous protein from the bacteria into the culture medium.
A number of recent advances have been made in bacterial protein expression, both relating to secretion and non-secretion systems. It has been observed, by a number of groups working in this field, that expression of soluble protein products is favoured by culturing the bacteria at 30.degree. C. or below.
For example, Cabilly (Gene, 85, p. 553-557, 1989) observed that expression of the Fd' fragment of an antibody directed against carcinoembryonic antigen (CEA) was improved at lower temperatures. This leads to a higher quantity of soluble heavy chain being recovered form the bacteria after lysis of the cells. The Fd' fragment was expressed with a complementary .kappa. light chain fragment in order to allow the formation of Fab fragments. A greater yield of active, soluble Fab fragments was obtained at 21.degree. C. and 30.degree. C. than at 37.degree. C.
Schein and Noteborn, Bio/Technology 6, p. 291-294, 1988, analysed the expression of three proteins, human interferon-.alpha.2 (IFN-.alpha.2), human interferon-.gamma. (IFN-.gamma.) and murine Mx protein at 37.degree. C. and at 23.degree.-30.degree. C. It was observed that proteins recovered from cell lysates were insoluble when the bacteria were grown at 37.degree. C. However, solubility was greatly increased by expression at 30.degree. C. The formation of insoluble protein is due to the aggregation of the heterologous polypeptide into inclusion bodies, a result of incorrect folding of the polypeptide chain.
The effect of temperature on proteins produced by secretion systems has been shown to be similar. Chalmers et al, in Applied and Environmental Microbiology, 56 (1), p. 104-111, 1990, demonstrated that both human interferon and .beta.-lactamase, both secreted proteins, produced in bacterial-cell culture were both more abundantly produced at 20.degree. C. than at 37.degree. C. Furthermore, the incidence of inclusion bodies was reduced at the lower temperature.
Chalmers et al conclude that for commercial production, as exemplified by chemostat experiments, culture at lower temperatures leads to more soluble proteins being produced.
Antibodies and antibody fragments, especially chimeric, recombinant or humanised derivatives thereof, are a class of proteins which it would be extremely desirable to be able to produce by recombinant DNA technology. By humanised antibodies, it is intended to refer to antibodies in which the constant regions are derived from human immunoglobulins, while at least the complementarity determining regions (CDRs) of the variable domains are derived from murine monoclonal immunoglobulins.
A number of improvements over natural immunoglobulins have been documented in the literature, which can only be put into practice by recombinant DNA technology. For instance, the production of CDR-grafted antibodies having CDRs from murine antibodies coupled to human framework regions can only be undertaken using a recombinant expression system. Furthermore, such systems are extremely useful for the production of antibody fragments which are not readily obtained by proteolytic cleavage, such as Fv fragments, and antibody fusions comprising an effector or reporter molecule attached to the antigen binding molecule.
Recombinant antibody fragments, whether they be entire antibodies, Fab, Fab', F(ab').sub.2 or Fv fragments, consist of heavy and light chain dimers. A recombinant expression system should therefore be capable of expressing both heavy and light chain genes in such a manner as to render the individual peptides capable of self-assembly into the final product. This has been a stumbling block for recombinant antibody production, and indeed attempts have been made to solve the problem. An example of this is the production of "single chain" Fv fragments, wherein the heavy and light chain polypeptides are physically joined together by a flexible linker group. These molecules avoid the problems of chain association between free heavy and light chain polypeptides.
This system is not necessary, however, for the production of larger antibody fragments such as Fabs, which comprise heavy and light constant region chains as well as heavy and light variable region chains. These fragments are large enough not to require coupling through a linker. For such applications it is desirable to express heavy and light chains separately in the same cell.
In order to facilitate correct assembly of heavy and light chains of antibody fragments, it is preferable to employ an expression system in which the chains are secreted into the periplasm of the host cell or into the culture medium rather than precipitated into the cell as inclusion bodies.
Dual Origin vectors (DUOV), for example as described in our U.S. Pat. No. 5,015,573, have been found to be particularly suitable for expressing antibody fragments. This has been shown to be the case, particularly when used in combination with protease-deficient bacterial host cells (see our International Patent Specification WO89/02465). The dual origin vector pAM1, (Wright et al., Gene, 49, p. 311, 1986), which comprises both pSC101 and colE1 replication functions, replicates at low copy number using the pSC101 replication functions at 30.degree. C. At this temperature, the colE1 replication functions, under the control of the .lambda. pR promoter, are tightly controlled by the c1857 repressor. Any foreign DNA inserted into the expression site of pAM1 is transcriptionally controlled by this being placed under the influence of the trp promoter, which is regulated by the host chromosomal trpR repressor.
The c1857 repressor is temperature sensitive, therefore increasing the temperature of the growth medium above 34.degree. C. leads to deregulation of the colE1 replication functions and increase in copy number from about 5 per cell to several hundred per cell. This causes the host trpR repressor to be titrated out, and transcription of foreign DNA from the trp promoter can take place.
However, secretion of antibody fragments from host cells transformed with DUOV vectors has not previously been attempted. In International Patent Specification WO89/02465 we describe a process for the expression of antibody fragments using DUOV vectors. However, the antibody fragments are expressed intracellularly and are precipitated as insoluble aggregates. These aggregates need to be solubilised by the use of chaotropic denaturants and/or other solubilisation techniques.